agronomy Article Phylogenetic Analyses of Rhizobia Isolated from Nodules of Lupinus angustifolius in Northern Tunisia Reveal Devosia sp. as a New Microsymbiont of Lupin Species Abdelhakim Msaddak 1,2, Luis Rey 3 , Juan Imperial 2, José Manuel Palacios 3 , Mohamed Mars 1 and José J. Pueyo 2,* 1 Laboratory of Biodiversity and Valorization of Arid Areas Bioresources, BVBAA, Faculty of Sciences, University of Gabès, Erriadh 6072, Tunisia; [email protected] (A.M.); [email protected] (M.M.) 2 Institute of Agricultural Sciences, ICA-CSIC, 28006 Madrid, Spain; [email protected] 3 Centre for Plant Biotechnology and Genomics, CBGP, UPM-INIA, Pozuelo de Alarcón, 28223 Madrid, Spain; [email protected] (L.R.); [email protected] (J.M.P.) * Correspondence: [email protected] Abstract: Thirty-two bacterial isolates were obtained from root nodules of Lupinus angustifolius growing in Northern Tunisia. Phylogenetic analyses based on recA and gyrB partial gene sequences grouped the strains into six clusters: four clusters belonged to the genus Bradyrhizobium (22 isolates), one to Microvirga (8 isolates) and one to Devosia (2 isolates), a genus that has not been previously reported to nodulate lupin. Representative strains of each group were further characterized. Multi- Locus Sequence Analysis (MLSA) based on recA and glnII gene sequences separated the strains within the genus Bradyrhizobium into four divergent clusters related to B. canariense, B. liaoningense, Citation: Msaddak, A.; Rey, L.; B. lupini, and B. algeriense, respectively. The latter might constitute a new Bradyrhizobium species. Imperial, J.; Palacios, J.M.; Mars, M.; The strains in the Microvirga cluster showed high identity with M. tunisiensis. The Devosia isolates Pueyo, J.J. Phylogenetic Analyses of Rhizobia Isolated from Nodules of might also represent a new species within this genus. An additional phylogenetic analysis based Lupinus angustifolius in Northern on the symbiotic gene nodC affiliated the strains to symbiovars genistearum, mediterranense, and Tunisia Reveal Devosia sp. as a New to a possibly new symbiovar. These results altogether contributed to the existing knowledge on Microsymbiont of Lupin Species. the genetic diversity of lupin-nodulating microsymbionts and revealed a likely new, fast-growing, Agronomy 2021, 11, 1510. https:// salt-tolerant rhizobial species within the genus Devosia as a potentially useful inoculant in agricultural doi.org/10.3390/agronomy11081510 practices or landscape restoration. Academic Editor: Alwyn Williams Keywords: Lupinus angustifolius; lupin; rhizobium; Devosia; Bradyrhizobium; Microvirga; microsym- biont; biodiversity; microbiota; Tunisia Received: 16 June 2021 Accepted: 26 July 2021 Published: 29 July 2021 1. Introduction Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in The Leguminosae or Fabaceae family includes more than 19,700 species, many of which published maps and institutional affil- are agronomically important [1]. The Genisteae tribe is presently considered to include iations. 618 species in 25 genera [2]. Lupinus is considered the largest Genisteae genus in terms of species number with around 270 species [3,4]. This genus may be divided into two groups, the first of which is centered in the Mediterranean Basin and the Canary Islands [5,6] and comprises 13 Old World species with smooth and rough seeds [3,7]. The second group includes the New World species [8]. The New World species number ranges from Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. 200 to 400 located in the Americas. Lupins are able to colonize very different environ- This article is an open access article ments and diverse ecosystems [9], including marginal impoverished or contaminated distributed under the terms and soils [10]. L. angustifolius, commonly named narrow leaf lupin and blue lupin, grows wild conditions of the Creative Commons in agricultural lands as well as in the wetlands of coastal plains in the Northern region Attribution (CC BY) license (https:// of Tunisia. creativecommons.org/licenses/by/ Lupins, partly due to their effective nitrogen-fixing symbiosis with different soil 4.0/). bacteria (collectively called rhizobia), have been grown since antiquity as an important Agronomy 2021, 11, 1510. https://doi.org/10.3390/agronomy11081510 https://www.mdpi.com/journal/agronomy Agronomy 2021, 11, 1510 2 of 17 pulse crop and as a green manure. Lupinus species have been studied from various perspectives as indicators of phosphorus and nitrogen in soils poor in nutrients, because of their dual efficacy in fixing atmospheric nitrogen and solubilizing soil phosphates [11], phytoremediation of polluted soils [12,13] and as medicinal plants [14].The high protein content of lupin seeds is similar to that of soybean, and lupin cultivation has been suggested as an alternative to soybean in Europe [15], as new sources are needed to meet the increasing demand for plant protein [16]. Rhizobia belong to the Alpha and Betaproteobacteria [17]. Legume-rhizobium sym- bioses provide a net input of nitrogen into the ecosystems. The great diversity of lupin species reflects a vast diversity of their nodulating rhizobia. Lupinus species are mainly nodulated by slow-growing bacteria that belong to the genus Bradyrhizobium. The major species are B. canariense, B. japonicum [18–21], B. elkanii [22], and B. valentinum [23]. Other fast-growing rhizobia species, such as Ochrobactrum lupini [24], Mesorhizobium loti [25], Phylobacterium trifolii [26] and Microvirga ssp. [27–29] can also effectively nodulate dif- ferent Lupinus spp. Recently, several microsymbionts isolated from L. albus growing in Tunisian soils were identified as species within the genera Agrobacterium, Rhizobium and Neorhizobium [30]. The genus Devosia was defined is a result of the reclassification of Pseudomonas riboflavin as Devosia riboflavina [31]. Devosia bacteria are aerobic, Gram-negative, rod-shaped or oval and they do not form spores. Twenty-one validated species have been identified within the genus (http://www.bacterio.net/devosia.html, accessed on 16 July 2020). Some Devosia species have been reported to nodulate legumes [32–34]. We have previously reported that L. angustifolius from Tunisia was nodulated by new lineages within the Bradyrhizobium and Microvirga genera [35], but, to the best of our knowledge, there are no reports on Devosia symbiotic nitrogen-fixing bacteria that nodulate Lupinus species. The main objective of the present paper was to further investigate and characterize the diversity of the rhizobia that nodulate L. angustifolius growing in different sites in Northern Tunisia. 2. Materials and Methods 2.1. Sampling Sites, Isolation of Bacterial Strains and Culture Conditions Effective nodules were collected from L. angustifolius roots growing wild or in soil collected from six different locations in Northern Tunisia: Mraissa, Takelsa, Borj Hfaied, Hammamet, Sejnane and Tabarka. The ecological and climatic characterization of the sampling sites is indicated in Table1. Nodules were surface-sterilized with 95% ethanol for 1 min, 25% sodium hypochlorite for 3 min, then rinsed several with sterile distilled water; they were individually crushed on sterile plates, and a loopful of nodule material was streaked onto yeast-mannitol agar (YMA) [36]. Plates were then incubated aerobically at 28 ◦C and colonies could be visualized after 4 to 10 days. Single colonies were streaked on fresh YMA plates. Pure isolates were maintained on YMA plates at 4 ◦C for temporary preservation, or stored in 20% (w/v) glycerol suspensions at −80 ◦C. The isolates used in this study are listed in Table1. 2.2. Nodulation and Cross-Inoculation Test The ability of all purified isolates to nodulate their original host was assessed. The isolates were also tested for nodulation on L. luteus, L. micranthus, L. mariae-josephae, Retama sphaerocarpa, Vigna unguiculata and Glycine max. Seed germination and plant inoculation were carried out as previously described [37]. Plants were grown under bacteriologically controlled conditions in a greenhouse for 3 to 8 weeks, depending on the legume host, with a 16.0/8.0 h light/dark photoperiod at 25/23 ◦C, and they were watered with sterile Jensen’s liquid medium once a week. Three uninoculated Leonard jars per legume were included as negative controls. Nodules were examined for number and inside color. Three replicates of each strain were used in the nodulation tests. Agronomy 2021, 11, 1510 3 of 17 Table 1. Designations and geographical origins of L. angustifolius endosymbionts in Northern Tunisia. Number Isolate Isolate Soil Bioclimatic Soil Site of Environment Code Code Number pH Stage Texture Isolates Takelsa 5 LanT 1, 3, 5, 6, 7 7.5 Area with orange trees semi-arid superior sandy Mraissa 5 LanM 4, 5, 6, 7, 9 8.0 Area planted with cereals semi-arid superior sandy Borj 2 LanB 5, 6 8.5 Area with olive trees semi-arid superior sandy Hfaiedh Hammamet 3 LanH 1, 2, 5 9.0 Semiurban area, next to the sea semi-arid superior sandy Sejnane 2 LanS 2, 5 7.5 Semiurban area humid clay 3, 4, 5, 6, 8, 9, 11, Tabarka 15 LanTb 12, 14, 15, 16, 17, 6.5 Area planted with cereals humid sandy 18, 19, 20 2.3. DNA Isolation, PCR Amplifications and Sequencing Bacterial DNA was obtained by the alkaline lysis method [38]. PCR was performed in a 25 µL volume containing DNA (5–10 ng), 2.5 µL of 10× PCR buffer containing magnesium chloride (Roche Applied Science), 10 µM of each primer, 10 mM of each dNTP, 1 µL DMSO and 1 U of Taq DNA polymerase (Roche Applied Science, Mannheim, Germany). PCR amplicons of genes coding for DNA recombination and repair protein RecA (recA), DNA gyrase subunit B (gyrB), 16S rRNA (rrs), chaperone protein DnaK (dnaK), glutamine synthetase 2 (glnII), and N-acetylglucosaminyl transferase (nodC) were obtained using previously described primers and conditions [37,39]. PCR amplifications of recA, 16S rRNA, glnII and nodC genes were performed and amplicons were sequenced as previously reported [37]. nifH was amplified by using the appropriate primers with PCR conditions previously described [34].